DEVELOPMENT OF A NOVEL BLOOD-BASED DIAGNOSTIC SYSTEM FOR MILD COGNITIVE IMPAIRMENT AND ALZHEIMER'S DISEASE USING CIRCULATING PEPTIDES

Discovering the signs of cognitive impairment at early stages followed by continuing appropriate intervention and treatment could delay the onset of dementia. Mass-spectrometry (MS)-based circulating peptide profiling have been suggested to be served as a blood-based biomarker, however, there is no convincing evidence for its diagnostic potential. In this study, we identified novel peptide biomarkers for mild cognitive impairment (MCI) and Alzheimer's disease (AD) using carefully collected blood samples from independent cohorts by differential peptidomics, and developed LC-MS/MS blood test using the peptide biomarkers for cognitive impairment. We performed screening of biomarker peptides by 2D-μLC-MALDI-TOF/MS using 300 serum samples in the longitudinal and cross-sectional studies from 3 independent cohorts, and validated them in multi-center clinical research. Next, we developed LC-MS/MS (SRM/MRM) assay and tested diagnostic potential of these biomarker peptides in assessment of cognitive impairment. Blinded serum samples from 930 subjects including MCI, AD and other neurological disorder were analyzed. The least absolute shrinkage and selection operator was used to evaluate the combination of multiple biomarkers. In discovery and validation phases, 14 peptide biomarkers for MCI and AD were identified by 2D-μLC-MALDI-TOF/MS, and, among them, 8 peptide biomarkers showed diagnostic potential for both MCI and AD in multi-center clinical studies by LC-MS/MS assay. The combinations of 3 biomarker peptides achieved an area under the curve of 0.86 (sensitivity 82%, specificity 80%) in MCI vs. NDC, and 0.89 (sensitivity 84%, specificity 84%) in AD vs. NDC, respectively. Individuals with lower MMSE scores had significantly elevated levels of the biomarker peptides. These peptides were derived from the proteins of complement system, fibrinolysis system and actin-binding protein family. Furthermore, levels of these peptides in the brain of definite AD and NDC subjects were analyzed by LC-MS/MS and immunochemistry against peptide-specific antibodies. The biomarker peptides were actually present in the brain and, in AD vs. NDC, differences of their levels in the hippocampus coincided with those in serum. The circulating peptides are potential biomarkers for MCI and AD in clinical use. The LC-MS/MS blood test system for peptide biomarkers is a powerful tool for early diagnostics for cognitive impairment.